Purification and characterization of glycerol‐3‐phosphate dehydrogenase ofSaccharomyces cerevisiae

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Abstract
The NAD-dependent glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate:NAD??? oxidoreductase; EC 1.1.1.8; G3P DHG) was purified 178-fold to homogeneity from Saceharomyces cerevisiue strain H44-3D by affinity- and ion-exchange chromatography, SDS-PAGE indicited that the enzyme had a molecular mass of approximately 42,000 (± 1.000) whereas a molecular mass of 68,000 was observed using gel filtration, implying that the enzyme may exist as a dimer, The pH optimum for the reduction or dihydroxyacetone phosphate (DHAP) was 7.6 and the enzyme had a pt of 7.4. NADPH will not subititute for NADH as coenzyme in the reduction of DHAP. The oxidation of glycerol-3-phosphate (G3P) occurs at 3% of the rate of DHAP reduction at pH 7.0. Apparent K m values obtained were 0.023 and 0.54 mM ror NADH and DHAP, respectively, NAD, fructose-1,6-bisphosphate (FBP), ATP and ADP inhibited G3P DHG activity, K 1 values obtained for NAD with NADH as variable substrate and FBP with DHAP as variable substrate were 0.93 and 4.8 mM, respectively.

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